We have successfully cloned and sequenced approximately 50% of the amyloid cDNA derived exclusively from Alzheimer's Disease (AD) brain messenger RNA (mRNA), and we are in the process of completing the entire molecule from the same source. The 1. 5 kb insert currently in use contains the A4 (beta-amyloid) polypeptide segment plus the flanking sequences; a second insert of at least 1.1 kb has been obtained that is upstream from the first insert and extends toward the 5' end. Therefore we propose to utilize cloned inserts from A4 and non-A4 regions for the following purposes: (A) We shall prepare suitable vectors to transfect neuronal glial and other cell types with the amyloid precursor to establish models for amyloid overproduction that may be relevant to the overaccumulation of amyloid in the AD brain. The transfected cells will be completely analyzed by in situ hybridization studies in our laboratories and by immunocytology using monoclonal antibodies to A4 and non-A4 regions (Project 3); the effects of amyloid overproduction on transcription, translation and cell survival will also be assessed. (B) We propose to establish transgenic mice using the cloned amyloid cDNA to obtain an animal model for amyloidosis that may be relevant to AD neuropathology. The mice will be analyzed by our molecular genetic techniques and by the immunologic, in situ hybridization, and molecular biological studies of other projects of the Program. (C) Using recombinant DNA methods, we shall generate various segments of the amyloid precursor protein for the in vitro proteolysis and posttranslational studies proposed in Project 5. (D) By similar procedures, we shall prepare radiolabeled probes from various segments of the precursor for in situ hybridization studies applied to the AD brain to answer questions on the molecular pathogenesis of AD (Project 2). (E) We shall use our existing cDNA libraries, as well as prepare additional libraries from sporadic and familial AD cases, to expedite the search for chromosome 21 products that are relevant to the etiology of AD, as proposed in Project 4. The data to be collected over the next several years is expected to provide new insights into amyloid production and accumulation in the AD brain and to suggest mechanisms to interrupt this pathological process at the molecular level.